Combinatorial approach to develop novel pre-therapeutic agents targeting virulence factors essential to clinically relevant pathogens

开发针对临床相关病原体必需毒力因子的新型治疗前药物的组合方法

• 批准号: 10681469
• 负责人: Hanh Ngoc Lam
• 金额: $ 24.07万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-09 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10681469
• 关键词: Active Sites; Affinity; Antibiotic Resistance; Antibiotic Therapy; Antibiotics; Aspartate; Bacteria; Binding; Binding Proteins; Biochemical; Biological; Biological Assay; Biology; C-terminal; Calorimetry; Cause of Death; Cell Death; Cell membrane; Cells; Cessation of life; Chemicals; Collaborations; Communicable Diseases; Computer Assisted; Consumption; Data; Development; Drug resistance; ESKAPE pathogens; Enzyme Activation; Fluorescent Probes; Foundations; Future; Goals; Gram-Negative Bacteria; Health; Health Care Costs; Host Defense Mechanism; Hybrids; Infection; Inflammation; Knowledge; Learning; Legal patent; Libraries; Medicine; Membrane; Mentors; Methods; Microbe; Molecular Target; Mus; N-substituted Glycines; Natural Products; Nosocomial Infections; Outcome; Pathogenesis; Pathogenicity; Pathology; Peptides; Peptoids; Periodicity; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phenotype; Phospholipase A2; Process; Productivity; Proteins; Pseudomembranous Colitis; Pseudomonas; Pseudomonas aeruginosa; Regulation; Research; Resistance; Resolution; Series; Serine; Serine Hydrolase; Specificity; Structure; Structure-Activity Relationship; Techniques; Therapeutic; Time; Titrations; Type III Secretion System Pathway; United States; Virulence; Virulence Factors; Virulent; Work; X-Ray Crystallography; acute infection; algorithm development; antibiotic resistant infections; antimicrobial; antimicrobial drug; appendage; clinically relevant; cofactor; combat; combinatorial; commensal microbes; cost; cost effective; design; drug candidate; drug discovery; extracellular; genetic approach; global health; healthcare-associated infections; high throughput screening; inhibitor; innovation; insight; lead optimization; macromolecule; marine; microbial; microbiome; microbiota; multidrug-resistant Pseudomonas aeruginosa; mutant; novel; novel strategies; novel therapeutics; pathogen; pathogenic bacteria; pressure; prevent; priority pathogen; screening; skills; small molecule; targeted treatment; therapeutic target; trait; virtual screening

Project Summary/Abstract Antibiotic resistance is a serious global health threat. Current antibiotics target essential bacterial processes and impose strong selective pressure for resistance. Upon antibiotic treatment, the healthy microbiota are reduced in both number and diversity, leading to serious health consequences such as Clostridium difficile colitis. Moreover, resistant traits can be transferred to other microbes, leading to the spread of antibiotic resistance. Using virulence blockers to target specific pathogenicity mechanisms, while leaving the microbiota intact, is a promising strategy to reduce resistance. This proposal will identify molecular target of a newly discovered the type III secretion system (T3SS) inhibitor, and explore their modes of action for further optimization and development. I will assess structure – activity relationship to optimize the T3SS inhibitors, cyclic pepeptomers, and use affinity based method to identify their molecular targets in Pseudomonas aeruginosa. Besides, target-based drug discovery offers the advantage of being low cost and less time consuming. With the availability of high-resolution structure and development of algorithms to predict binding affinity and poses of small molecules to its protein target, virtual screening can provide lead for optimization. ExoU, an effector with phospholipase A2 activity, is the major effector in P. aeruginosa, one of six ESKAPE pathogens which cause the majority of nosocomial infections in the U.S. and “escape” antimicrobial drugs. ExoU has a serine/aspartate catalytic dyad and a separate cofactor-binding domain required for activation of the enzyme. ExoU is highly toxic, associated with acute infection, antibiotic resistance and severe outcome in patents. Delay ExoU expression can increase mice survival. Thus we set out to find ExoU inhibitors as a strategy to treat acute infection and reduce resistance. We will identify ExoU inhibitors that 1) inhibit enzymatic activity by targeting its catalytic residue serine, or 2) bind to the membrane localization domain which will prevent ExoU's activation by virtual screening. The inhibitors that show binding affinity to ExoU in isothermal titration calorimetry assay, and prevent cell death caused by ExoU will be chosen for optimization. Structure of inhibitor-bound proteins will be solved using X-ray crystallography. I believe that my team of mentors (Drs. Stone and Ottemann), advisors (Dr. Rubin, expert in X-ray crystallography; Dr. Jacobson, expert in computer- aided drug discovery) and collaborators (Dr. Lokey, an expert in macromolecule synthesis, Drs. Crews and Linington, natural product chemists) will provide me support to successfully carry out this project. With the biochemical techniques I will learn, the structures and new inhibitors I will obtain in the K99 phase, I will then collaborate with Dr. Shaw (medicinal chemist) and Dr. Jacobson to optimize the candidate hits in my independent phase. This project extends my skill set in biochemical methods and has the potential to provide substantial momentum towards drug discovery, and development. These will serve as the foundation of a R01 proposal to be prepared upon the completion of the main stages of this research plan.

项目总结/摘要 抗生素耐药性是一个严重的全球健康威胁。目前的抗生素靶向基本的细菌过程 并施加强大的选择压力来抵抗。在抗生素治疗后，健康的微生物群 数量和多样性减少，导致严重的健康后果，如艰难梭菌 结肠炎此外，耐药特性可以转移到其他微生物，导致抗生素的传播 阻力使用毒力阻断剂靶向特定的致病机制，同时使微生物群 是一个很有希望的减少耐药性的策略。这一建议将确定一个新的分子靶点 发现了III型分泌系统（T3 SS）抑制剂，并探索其作用机制，以进一步研究其作用机制。 优化和发展。通过构效关系的评价优化T3 SS抑制剂， 环肽异构体，并使用基于亲和力的方法来鉴定它们在假单胞菌中的分子靶标 铜绿。此外，基于靶点的药物发现具有成本低、时间短的优点 消耗。随着高分辨率结构的可用性和预测结合的算法的发展 小分子与其蛋白质靶点的亲和力和位姿，虚拟筛选可以为优化提供线索。 ExoU是一种具有磷脂酶A2活性的效应子，是铜绿假单胞菌中的主要效应子， 病原体导致美国大部分医院感染，并“逃避”抗微生物药物。 ExoU具有丝氨酸/天冬氨酸催化二联体和激活细胞所需的单独的辅因子结合结构域。 酶ExoU具有高毒性，与急性感染、抗生素耐药性和严重结局相关。 专利延迟ExoU表达可以增加小鼠的存活率。因此，我们开始寻找ExoU抑制剂作为一种 治疗急性感染和减少耐药性战略。我们将鉴定ExoU抑制剂，其1）抑制酶促 活性通过靶向其催化残基丝氨酸，或2）结合到膜定位结构域， 通过虚拟屏蔽阻止ExoU激活。在等温条件下显示出与ExoU结合亲和力的抑制剂 滴定量热法测定，并防止由ExoU引起的细胞死亡将被选择用于优化。结构 将使用X射线晶体学来解析与靶蛋白结合的蛋白质。我相信我的导师团队（博士。 斯通和奥特曼），顾问（鲁宾博士，X射线晶体学专家;雅各布森博士，计算机专家- 辅助药物发现）和合作者（Lokey博士，大分子合成专家，Crews博士和 Linington，天然产物化学家）将为我提供支持，以成功地开展这一项目。与 我将学习的生化技术，我将在K99阶段获得的结构和新的抑制剂，我将 与肖博士（药物化学家）和雅各布森博士合作，优化我的候选命中率。 独立阶段。这个项目扩展了我在生物化学方法方面的技能，并有可能提供 药物发现和开发的巨大势头。这些将作为R 01的基础 在完成本研究计划的主要阶段后编写的建议。